Food supplemented with a carnitine, suitable for stimulating the biosynthesis of polyunsaturated fatty acids from the saturated fatty acids contained in the food

ABSTRACT

A food selected from the group comprising milk and dairy products derived from milk, comprising a carnitine in an effective amount to stimulate, through the natural fatty acid metabolic processes that take place in a consumer of said food, the synthesis of polyunsaturated fatty acids from the saturated fatty acids originally contained in the food, the synthesis of polyunsaturated fatty acid from the saturated fatty acids originally contained in the food.

This application is the U.S. national phase of international applicationPCT/IT00/00497 filed 30 Nov. 2000, which designated the U.S.

The present invention relates to milk and food stuffs derived from theprocessing of milk (i.e. dairy products) supplemented with an agentsuitable for stimulating, through the natural metabolic processes thattake place in the body, the synthesis of polyunsaturated fatty acidsstarting from the saturated fatty acids originally contained in theabove-mentioned foods.

In the context of the present invention what is meant by “milk”, whichby definition is the integral product of a complete, uninterruptedmilking of a healthy, well nourished milk-bearing female, obtained bymanual or mechanical milking, is not only cow's milk and the skimmedmilk, delactosed milk, powder milk and condensed milk derived from it,but also cow-buffalo's milk, goat's milk and sheep's milk.

Therefore, in the context of the present invention, what is meant by“food stuffs derived from the processing of milk” are not only butter,cream, cottage cheese, yoghurt, kefir, milk cheese (i.e. cow's milkmozzarella) and fresh and ripened cheeses (such as, for example, granapadano and Parmesan cheese) derived from the processing of cow's milk,but also cheese products derived from the processing of cow-buffalo's,goat's and sheep's milk, such as, for example, buffalo milk cheese ormozzarella, provola, ewe cheese or Pecorino (e.g. Roman Pecorino,Sicilian Pecorino), Sardinian milk cheese, Urbino sheep's milk cheese,blue cheese, etc.

Fresh milk, i.e. milk from animals that have just been milked, consistsessentially, with more or less minor variations of the variouscomponents from one animal species to another, of 87-88% water, 4.5-4.8%sugars, 3.5-7.5% lipids, 3.2-6% proteins and traces of mineral salts andvitamins.

Milk lipids consist substantially in fatty acid triglycerides whichconstitute 96-99% of total lipids (the remaining fraction being mainlycomposed of diglycerides, phospholipids, sterols and cerebrosides).

Fatty acids are classified as saturated and unsaturated on the basis ofthe presence or otherwise of double bonds in their chain. Saturatedfatty acids (whose synthesis is catalysed by different enzyme systemssuch as acetyl-CoA-carboxylase, fatty acid synthetase and citratesynthetase) do not contain double bonds, while the unsaturated fattyacids present one or more double bonds between the carboxyl and theterminal methyl at the opposite end of the chain. Whereas a role inenergy metabolism is mainly attributed to the saturated fatty acids, theunsaturated fatty acids also have important biological functions asstructural components of the membranes, as precursors of theeicosanoids, such as prostaglandins and leukotrienes, and as cholesteroltransport agents.

Starting from acetyl-coenzyme A, the body, by means of a synthetaseenzyme system, is capable of synthesizing fatty acids with up to 16carbon atoms, elongating their chain and desaturating them at themicrosomal level.

This desaturation does not occur in man between the methyl and seventhcarbon atom proceeding. towards the carboxyl, and the essentiality ofpolyunsaturated fatty acids omega-3 (n3) and omega 6 (n6) is dueprecisely to this inability.

Two important polyunsaturated essential fatty acids such as linoleicacid (c18:2n6) and linolenic acid (c18:3n3) therefore have to beingested with the diet.

The table here below presents the fatty acid percentage composition ofthe lipids of cow's milk.

Cow's milk lipids Fatty acids I.M.* R** Saturated fatty acids Butyric4:0 3.3 2.3-5.2 Caproic 6:0 2.1 1.4-4.0 Caprylic 8:0 1.4 0.9-2.2 Capric10:0 3.0 2.2-4.2 Lauric 12:0 3.6 2.4-4.6 Myristic 14:0 11.5  8.4-13.6Palmitic 16:0 30.8 24.0-36.9 Stearic 18:0 9.5  6.6-13.2 Arachic 20:0Other 2.4 Monounsaturated fatty acids Myristoleic 14:1 1.9 1.4-2.2Palmitoleic 16:1 2.8 2.2-3.3 Oleic 18:1 22.5 25.9-18.8 Other 1.3Polyunsaturated fatty acids Linoleic 18:2 2.3 1.4-3.6 Linolenic 18:3 1.50.6-2.8 *Indicative mean **Range

Palmitic acid is the most important fatty acid produced by most of thebiosynthesis systems, and other saturated or unsaturated long-chainfatty acids can be derived from it.

The fatty acid desaturation site for the formation of unsaturated fattyacids is located in the microsomes and the introduction of a double bondin a preformed fatty acid requires the intervention of molecular oxygenas an electron acceptor. This system, which is called mixed-functionoxygenase, in the presence of O₂ and NADHP, catalyzes the desaturationof preformed fatty acids in the form of acyl-CoA.

Undergoing various processes of elongation and desaturation, palmiticacid plays a central role in the formation of unsaturated fatty acids.

The fats contained in milk and its derivatives also undergo themetabolism common to all fatty acids, in which the presence ofL-carnitine is of fundamental importance for their utilisation.

The role of L-carnitine and alkanoyl L-carnitines, particularly ofacetyl L-carnitine is, in fact, essential in the processes of fatty acidbeta-oxidation which takes place above all in the mitochondria withprior transformation to the thioesters of Coenzyme A catalyzed byacyl-CoA-thiokinase. The transport of acyl-CoAs from the activationsites to the oxidation sites is carried out by L-carnitine by means ofan enzyme, acyl-CoA carnitine acyltransferase, located on the innermembrane of the mitochondria which is permeable to the carnitine estersbut not to the acyl-CoAs, CoASH or free L-carnitine.

As can be seen in the table above, the quantity of essential fatty acidspresent in cow's milk is very low: 2.3% for linoleic acid and 1.5% forlinolenic acid, as against 23% for oleic acid, 30% for palmitic acid,11% for myristic acid and 9.5 for stearic acid.

These low concentrations of the essential fatty acids, linoleic andlinolenic acid, are totally inadequate for balanced nutrition based onthe intake of milk, as is the case above all in infant children, butalso in the elderly and in convalescent subjects. Nor, obviously, canthe intake of essential fatty acids be increased by introducing into thediet products (dairy products) obtained from the processing of milk.

In the past, milk was produced supplemented with factors which are notpresent or are present in inadequate amounts in normal cow's milk (e.g.vitamins, nitrogenous substances, etc.) bearing in mind not only theparticular dietetic requirements of infants, but also those of elderlysubjects, sick people and convalescent subjects. More recently, in thewake of the interest aroused by extensive clinical studies which haveproved the therapeutic efficacy, in cardiovascular disease, ofpolyunsaturated fatty acids of the omega-3 and omega-6 series, which areparticularly present in the fish-rich diets of certain Nordic peoples,types of milk supplemented with omega-3 fatty acids have been marketedfor general consumption.

The addition of these fatty acids to the milk, however, entails a numberof technological problems due to the need to obtain a product whichmaintains the characteristics of stability and conservability and, aboveall, the agreeable organoleptic properties of natural milk. It is wellknown, in fact, that the omega-3 series fatty acids such aseicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are extractedfrom fish oil and present a substantial tendency to oxidation in airwith the result that the milk turns rancid, converting to oxidizedsideproducts with an irritating and distinctly disagreeable odour andtaste.

It has now surprisingly been found that the addition of a carnitine (aterm which will be defined here below) to the above-mentioned milk anddairy products powerfully stimulates, through the natural metabolism offatty acids that occurs in a consumer of such foods, the synthesis ofpolyunsaturated fatty acids starting from the saturated fatty acidsoriginally contained in such foods.

The object of the present invention is, therefore, a food selected fromthe group comprising milk and dairy products, characterized in that itcomprises a carnitine selected from the group comprising of L-carnitine,acetyl L-carnitine, propionyl L-carnitine or their pharmacologicallyacceptable salts, or mixtures thereof, in an effective amount forstimulating the synthesis of polyunsaturated fatty acids from thesaturated fatty acids originally contained in said food, in the courseof the natural metabolic processes that take place in a consumer of saidfood.

The milk can be selected from cow's milk (or skimmed milk, delactosedmilk, powder milk or condensed milk obtained from cow's milk),cow-buffalo milk, goat's milk or sheep's milk. The dairy productsobtained from the above-mentioned milks include products such as butter,cream, cottage cheese, yoghurt, kefir, milk cheese or cow's milkmozzarella, fresh and ripened cheeses (e.g. grana padano or Parmesancheese), cow-buffalo milk cheese or mozzarella, provola, ewe cheese orPecorino, Roman Pecorino, Sicilian Pecorino, Sardinian milk cheese,Urbino sheep's milk cheese and the like.

The amount of L-carnitine, acetyl L-carnitine and propionyl L-carnitine(alone or in various mixtures) added to the milk or dairy productsobtained from milk may vary within broad limits owing to the substantialnon-toxicity of these compounds. In the case of “milk”, the amount mayrange from 400 mg to 8,000 mg/L of food product, and preferably from1,000 mg to 4,000 mg/L.

In the case of butter, yoghurt, kefir and both fresh and ripenedcheeses, such amount may range from 500 mg 70 9,000 mg/kg of foodproducts, and preferably from 1,500 to 4,500 mg/kg of food product.

Both the “milk” and the products derived from milk processing can besupplemented with other substances selected from the group comprisingvitamins (e.g. vitamin E, vitamin C, vitamin B₆), coenzymes, mineralsubstances, amino acids and antioxidants, polyphenols (of grapes),catechins (of tea), anthocyanins, selenium (methionine), calcium saltsand the like.

A number of pharmacological tests illustrating the invention describedherein are reported here below.

Plasma Lipid Tests in Animals on a Diet Consisting of Milk Alone or MilkPlus Carnitines

For these tests a batch of New Zealand male rabbits with a mean bodyweight of 3.7 kg was used, which, after a period of acclimatisation on astandard diet for eight days, were divided into different groups. Onegroup received standard diet alone, while a second group receivedunskimmed milk mixed with pellets of the normal diet whose finalcomposition was as follows: 4% fats, 24% proteins, 58% carbohydrates,0.7% fibre, 10% water, 3% minerals and 1% vitamins.

Another group of animals was administered a milk diet supplemented withL-carnitine, acetyl L-carnitine or propionyl L-carnitine in amounts suchthat, on the basis of the food intake, the daily administration was 200mg/kg body weight of L-carnitine, acetyl L-carnitine or propionylL-carnitine.

After four weeks of treatment, blood samples were taken from themarginal vein of the ear and the plasma lipids obtained were subjectedto measurement of the percentages of both saturated fatty acids andpolyunsaturated fatty acids. The percentage amounts of unsaturatedomega-3 fatty acids present in liver samples taken from the same animalswere also measured. While total lipids were measured according to themethod described by Schanfeld (Schanfeld G., J. Clin. Invest., 226:497,1957) and Folch (Folch J., J. Biol. Chem., 226:497, 1957), thetriglycerides and phospholipids were measured according to the methoddescribed by Terstra (Terstra A. H. M., Anal. Biochem., 11:149, 1981)and the n-3 and n-6 fatty acids according to the method described byNestel (Nestel P. J., Ann. Rev. Nutr., 10:149, 1990) and Cevec (CevecG., Biochemistry, 30:7:7186, 1991).

The data reported in Table 1 demonstrate that the milk diet does notimprove the ratio of polyunsaturated to saturated fatty acids, whichappears unchanged or slightly worsened as compared to controls. In thoseanimals in which carnitines were administered together with the samemilk diet, the ratio was distinctly improved due to an increase inpolyunsaturated fatty acids, and this was particularly evident followingadministration of acetyl L-carnitine and propionyl L-carnitine.

This new particular and unexpected activity of the carnitines added tothe milk was also evident in the examinations performed on the liverspecimens taken from the animals on the different diets. The data inTable 2, in fact, show a surprising increase in n-3 fatty acids,resulting in the improvement of the ratio of n-3 to n-6 fatty acids. Inthese tests, too, propionyl L-carnitine proved to be the most activecompound.

TABLE 1 P/S (polyunsaturated to saturated fatty acids) ratio in basalconditions and after 30 days on various diets. day 0 day 30 Standarddiet 0.55 0.52 Milk diet 0.59 0.47 Milk diet + L-carnitine 0.52 0.72Milk diet + acetyl L-carnitine 0.56 0.85 Milk diet + propionylL-carnitine 0.55 0.88

TABLE 2 n-3 and n-6 fatty acids present in hepatic phospholipids.Percentage values of total lipids at baseline and after 30 days on diet.Phospholipid baseline values n-6 = 20.5 n-3 = 76.9 n-6:n-3 ratio = 1.215Phospholipid values after 30 days n-6 = 13.5 n-3 = 35.6 n-6:n-3 ratio =0.38

The milks supplemented with carnitine according to the present inventionare not to be confused, either as regards composition or as regardsaims, with the “infant formulas”, possibly containing carnitine, alreadyon the market or described in the literature. The infant formulas areartificial milks in which the manufacturer's primary aim is to reproducethe composition of mother's milk (see, for example, U.S. Pat. Nos.3,542,560, 4,282,265, 4,614,663, 4,721,626 and 4,879,131). Morerecently, infant formulas have been proposed, supplemented withsubstances suitable to prevent or cure even severe diseases typical ofneonates or suckling infants. For example, U.S. Pat. No. 5,686,491discloses an infant formula containing 2.5-3.5 g of proteins/kg bodyweight, carbohydrates, lipids and at least 50-150 mg of L-carnitine/kgbody weight, in which the carbohydrate:lipid ratio is equal to orgreater than 60:40. This infant formula is particularly suitable for thenutrition of suckling infants suffering from fatty acid catabolismdisorders such as medium-chain acyl-CoA dehydrogenase deficiency (MCAD),long-chain acyl-CoA dehydrogenase deficiency (LCAD), short-chainacyl-CoA dehydrogenase deficiency (SCAD) and multiple-chain acyl-CoAdehydrogenase deficiency (MADD) and for the prevention of cases ofsudden infant death syndrome (SIDS) and growth failure syndrome (FITS).

The marked differences in composition, destination and aims between theinfant formulas and the milks according to the present invention aretherefore evident.

Also evident are the advantages which can be achieved with the foodsaccording to the invention, which, after being ingested and inconjunction with the natural metabolic processes of the consumer, makeit possible to increase the endogenous amount of polyunsaturated fattyacids with an attendant reduction in the saturated fatty acids in whichthese foods, especially butter and ripened cheeses, are rich. This isparticularly significant in view of the growing attention which is beingjustifiably accorded to diet, also by perfectly healthy individuals, andto the care taken to avoid foods with a high content of saturated fattyacids for the purposes of preventing various diseases including mostnotably metabolic disorders and cardiovascular diseases.

The present invention makes available dairy products which, whilst fullyconserving their nutritional and organoleptic properties, are equivalentto products with a higher content of unsaturated fatty acids and a lowercontent of saturated fatty acids, thus assuring safer and widerconsumption.

Furthermore, from the technological standpoint, the addition ofcarnitine to milk and its derivatives presents none of theabove-mentioned operational difficulties caused by the addition to milkof substances which easily turn the milk rancid, such as fatty acids ofthe omega-3 series, with the consequent hazard of altering theorganoleptic properties of the resulting food product which, despitesuch addition, maintains its original disadvantageously high content ofsaturated fatty acids.

Illustrative, non-limiting examples of compositions according to theinvention are. reported hereinbelow.

1) Whole cow's milk ml 100 L-carnitine mg 100 2) Skimmed or partiallyskimmed cow's milk ml 100 L-carnitine mg 50 3) Whole cow's milk ml 100Acetyl L-carnitine mg 100 4) Skimmed or partially skimmed cow's milk ml100 Acetyl L-carnitine mg 50 5) Whole cow's milk ml 100 PropionylL-carnitine mg 100 6) Skimmed or partially skimmed cow's milk ml 100Propionyl L-carnitine mg 50 7) Whole cow's milk ml 100 L-carnitine mg 50Acetyl L-carnitine mg 50 Propionyl L-carnitine mg 50 8) Skimmed orpartially skimmed cow's milk ml 100 L-carnitine mg 25 Acetyl L-carnitinemg 25 Propionyl L-carnitine mg 25 9) Yoghurt g 100 L-carnitine mg 50Acetyl L-carnitine mg 50 Propionyl L-carnitine mg 50 10)  Butter g 100L-carnitine mg 100 Acetyl L-carnitine mg 100 Propionyl L-carnitine mg100 11)  Whole cow's milk ml 100 L-carnitine mg 100 Acetyl L-carnitinemg 100 Propionyl L-carnitine mg 100 Vit. E mg 2 Vit. C mg 30 Vit. B₆ mg0.5 12)  Whole cow's milk ml 100 L-carnitine mg 50 Acetyl L-carnitine mg50 Propionyl L-carnitine mg 50 Grape's polyphenols mg 100 Catechins(derived from tea) mg 100 Calcium mg 100 13)  Yoghurt g 100 L-carnitinemg 50 Acetyl L-carnitine mg 50 Propionyl L-carnitine mg 50 Polyphenolsmg 100 Catechins mg 100 Anthocyanins mg 20 Selenium methionine μg 50

1. A food consisting of a dairy product derived from milk comprising acarnitine selected from the group consisting of L-carnitine, acetylL-carnitine, propionyl L-carnitine, a mixture thereof and apharmacologically acceptable salt of the carnitine, in an effectiveamount of from 400 mg to 9,000 mg/L or mg/kg of food product tostimulate upon ingestion, through the natural fatty acid metabolismprocesses that take place in a consumer of said food, the synthesis ofpolyunsaturated fatty acids from the saturated fatty acids originallycontained in the food.
 2. The food of claim 1, wherein the food is adairy product derived from the processing of cow's milk, cow-buffalo'smilk, goat's milk or sheep's milk.
 3. The food of claim 2, wherein thefood is a dairy product and is derived from the processing of cow's milkand is selected from the group consisting of butter, cream, cottagecheese , yoghurt, kefir, mozzarella cheese, fresh cheese, ripe cheese,grana padano and parmesan cheese.
 4. The food of claim 2, wherein thefood is a dairy product derived from the processing of cow-buffalo'smilk, goat's milk or sheep's milk and is selected from the groupconsisting of cow-buffalo's mozzarella cheese, provola, goat's milkcheese, ewe's milk, Roman pecorino, Sicilian pecorino, Sardinian cheeseand caciotta d'Urbino.
 5. The food of claim 2, comprising from 500 to9,000 mg/kg of a carnitine selected from the group consisting ofL-carnitine, acetyl L-carnitine, propionyl L-carnitine and a mixturethereof or a pharmacologically acceptable salt of the carnitine.
 6. Thefood of claim 5, comprising from 1,500 to 4,500 mg/kg of a carnitineselected from the group consisting of L-carnitine, acetyl L-carnitine,propionyl L-carnitine or a mixture thereof or a pharmacologicallyacceptable salt of the carnitine.
 7. The food of claim 1, furthercomprising vitamins, coenzymes, mineral substances, amino acids andantioxidants.
 8. A method of stimulating in a consumer of a foodconsisting of a dairy product derived from milk, through the naturalfatty acid metabolism processes of said consumer, the synthesis ofpolyunsaturated fatty acids from the saturated fatty acids originallycontained in the food, which process comprises administering to theconsumer a food consisting of dairy products derived from milk to whichhas been added a carnitine selected from the group consisting ofL-carnitine, acetyl L-carnitine, propionyl L-carnitine, a mixturethereof and a pharmacologically acceptable salt of the carnitine, in aneffective amount to stimulate said synthesis.
 9. The method of claim 8,wherein the food is a dairy product and the quantity added to the dairyproduct is 500-9,000 mg/kg of the dairy product.